Identification and DNA Barcoding of a New Sillago Species in Beihai and Zhanjiang, China, with a Key to Related Species

YU Zhengsen, GUO Ting, XIAO Jiaguang, SONG Na, and GAO Tianxiang

Identification and DNA Barcoding of a NewSpecies in Beihai and Zhanjiang, China, with a Key to Related Species

YU Zhengsen1), GUO Ting1), XIAO Jiaguang2), SONG Na1), and GAO Tianxiang3), *

1)Fishery College, Ocean University of China, Qingdao 266003, China 2) Laboratory of Marine Biology and Ecology, Third Institute of Oceanography, Ministry of Natural Resources,Xiamen 361005, China 3) Fishery College, Zhejiang Ocean University, Zhoushan 316022, China

A newspecies,sp. n., is identified based on 127 specimens collected from the southern coast of China. We compared the morphological characters betweenand all other 11species with two posterior extensions on the swim bladder. The new species is likein the countable characters and color pattern, but is different from the latter by the distinct swim bladders. The swim bladder of.is without lateral process. The posterior sub-extensions of anterolateral extensions are unique with some dendritic or sometimes stunted blind tubule, which are unilateral and outward, extending along the abdominal, and are about one-third to half of the body of swim bladder in length. But the swim bladder of.with 8–10 lateral processes, the posterior sub-extensions of anterolateral extensions are kinky, long and complicated, extending along the abdominal wall below the peritoneum to the base of posterior extensions..can be distinguished from other species in this group by color pattern, meristic, and morphometric characters. Moreover, the results of genetic analysis using sequences of the mitochondrial cytochrome c oxidase subunit I () fragment show significant interspecific-level genetic distances (0.159–0.231) betweenand 8 congeners in the group, which also support the validity of new species. We also provide a distribution map and a key of the related species.

new species;sp. n.; morphological characteristics; DNA barcoding; taxonomy

1 Introduction

Sillaginids are small to moderate sized Indo-West Paci- fic fishes, commonly inhabiting in inshore waters of sandy substrates or estuarine areas of rivers (McKay, 1992). The family consists of thirty-six species (Fricke, 2020), including five new described species during last ten years:.Kaga, Imamura & Nakaya, 2010;.Gao & Xue, 2011;.Golani, Fricke & Tikochinski, 2014;.Gao & Xiao, 2016; and.Panh- war, 2017 (Kaga, 2010; Gao, 2011; Golani,2014; Xiao, 2016; Panhwar, 2018). Furthermore, another unnamed species,sp., was recorded from the southern coast of China (Xiao, 2018).

Sillaginids are easily identified as a family by their si- milarity of external morphological characters (Sano and Mochizuki, 1984; McKay, 1992), but hard in specific iden- tification. The external morphological similarity has made many true species be concealed in the synonymy of those wide-ranging species. For instance, separate identities ofthe three trumpeter sillaginids,,, and, were only recognized in 1985 and are ele- vated to full species by McKay in 1992 (McKay, 1985, 1992). In addition,, an Indo-West Pacific species, exhibits many cryptic lineages across its distribu- tion range (McKay, 1992). The morphology of the swim bladder has been proved to be effective in distinguishingspecies (McKay, 1985, 1992; Xiao, 2018). Accord- ing to this character, McKay (1985) recognized three sub- genera of:Fowler 1933 (swim blad- der reduced, no duct-like process);Cuvier 1817 (swim bladder divided posteriorly into two tapering ex- tensions, duct-like process present); and(Mc- Kay, 1985) (swim bladder with a single posterior exten- sion and the duct-like process). However, it may be still difficult to distinguish close-related species in this groups based on morphology only. In the past decades, the utility of DNA barcoding on species delimitation and identifica- tion has been demonstrated by numerous studies (Hebert., 2003; Ward., 2005; Burns., 2008; Locke., 2010). And DNA barcoding is particularly useful in recovering possible new species when morphological dif-ferentiation within groups is imperceptible (Gao, 2011; Golani, 2014; Xiao, 2016).

In the present study, we delimited a newspeciesfrom two locations of southern coast of China through mor- phological and genetic comparisons. A phylogenetic tree was reconstructed to help distinguish species. We also pro- vided a distribution map and a key ofspecies with two posterior extensions on the swim bladder.

2 Materials and Methods

2.1 Materials Examined

Specimens of the new species were collected from Zhan- jiang and Beihai, China. Institutional abbreviations follow Fricke and Eschmeyer (2020) except for Fishery Ecology & Marine Biodiversity Laboratory, Fishery College, Zhe- jiang Ocean University (FEMBL).

Comparative materials (see Table 1 for number of spe- cimens).: BPBM 36393, holotype, 148.6mm SL, Salalah fish market, Oman, photograph.: NMMB-P13995, 174.2mm SL, Phant Thiet, Viet- nam, photograph; USNM FIN34286, 151mm SL, Salalah, Oman, February, 1993, photograph.: BMNH 1976.11.17.1–2, 2, paratypes, 81.0–101.0mm SL, photographs.: FAMB-01-1611, holotype, 160mm TL, northern Arabian Sea coast of Pakistan, pho- tograph.: FEMBL 150001, holotype, 182.2mm SL, Xiamen, China, December 2013; FEMBL150002–150016: 15, paratypes, 173.0–203.3mmSL, Xiamen, Chi-na, December 2013; FEMBL 150017–150019: 3, para- types, 146.2–167.6mmSL, Changhua County, Taiwan, China, July 2014; FEMBL 150020–150039: 20, paratypes, 182.0–216.0mm SL, Dongshan, China, April 2014.: FEMBL 150101–150108: 8, Zhuhai, No- vember 2014; FEMBL 021573–021583: 11, Bahrain, Oc- tober 2014; FEMBL 021220–021224: 5, Karachi, January to February 2016; FEMBL020918–020927: 10, PhuketIsland, March 2015.: OUC_FEL100348, ho-lotype, 122.79mm SL, Wenzhou, China, June 2009; OUC_ FEL174001–174030: 30, 101.8–162.5mm SL, Rushan, Shandong, China, August, 2014; OUC_FEL100362– 100388, 100403: 28, 94.36–134.17mm SL, near Dong- ying, Shandong, China, September, 2009.: HUJF 7134, holotype, 137mm SL, Abu Zanima, Egypt,Gulf of Suez, Red Sea, photograph.sp.: FEMBL 021255–021283: 29, 127.8–155.6mm SL, Xiamen, China, December, 2015; FEMB L021284, 167.6mm SL, Zhang- hua, Taiwan, China, July 2014.

Table 1 Number of specimens used for morphological and genetic comparisons in this study

2.2 Morphological Analysis

The genus and species classification followed McKay (1985) unless otherwise noted. The terminology of appen- dages of the swim bladder followed Shao. (1986) and Kaga and Ho (2012). In the descriptive section, holotype data were given first, followed by paratypes data in paren- theses. Specimen length was standard length, SL. Head length was abbreviated as HL. Measurements were made with dial calipers and the dividers were accurate to 0.1mm and weights to the nearest 0.1g. The definition of the modified vertebrae followed McKay (1992). The swim bladder anatomy observations were performed after dis- section of 20 paratypes. Specimens of the new species were preserved in alcohol.

After measurements and counts, both sagittaes were ta- ken from each specimen. Each was placed in a 1.5-mL plas- tic tube containing distilled water and soaked for 1h. Fol- lowed by ultrasonic cleaning for about 30min, the otoliths were baked in an oven for 24h at 55℃ until a constant mass to the nearest 0.01mg was achieved. The otolith’s digital images were taken with a Nikon SMZ800 microscope equip- ped with a Nikon digital sight DS-Fi1 (Tokyo, Japan).

2.3 Genetic Analysis

To analyze genetic differences between new species and otherspecies, mitochondrial (mt) DNA cytochro- me oxidase subunit I () fragments were amplified basedon the method of Ward. (2005). Genomic DNA ex- traction and polymerase chain reaction (PCR) followed Gao. (2011). Thefragment amplification primer used in PCR reaction system is L5956-(5’-CACAAA GACATTGGCACCCT-3’) and H6558-(5’-CCTCCT GCAGGGTCAAAGAA-3’) (Inoue., 2001).

sequences produced in the present study were sub- mitted to GenBank with the following accession numbers (see Table 1):.(MH 093482–MH093490),.(MF571945, MF571947, KU051787 and KU 051788), andsp. (MG571458, MG571455, MG 911029, MG911030, KU 051812). Thesequences of other species were downloaded from GenBank with the following accession numbers (see Table 1):.(KM 350229–KM350232),.(KU051872, KU051 873, KU051879, KU051886 and KU051887),.(KU 051813, KU051819, KU051857, KU051803 and KU051 881),.(KU052012, KU052017, KU052023, KU 052025 and KU052029),.(FJ155362–FJ155 364), and.(HQ389247–HQ389249). In to- tal, the presently available ninespecies with two posterior extensions of the swim bladder were used for ge-netic comparison. Sequences were then checked and align- ed using the DNASTAR software (DNASTAR Inc., Ma- dison, WI. USA). MEGA 5.0 (Tamura., 2011) was used to reckon the genetic distances and reconstruct a Neighbor-joining (NJ) tree under the Kimura 2-parameter (K2P) model usingas the outgroup.

3 Results

3.1 Taxonomy

Family Sillaginidae Richardson, 1846

GenusCuvier, 1817

()sp. n. Gao, Xiao & Guo (Fig.1)

Fig.1 Sillagoparasihama sp. n. Gao, Xiao & Guo, FEM BL150036, holotype, 132.5mm SL, Zhanjiang, China.

Holotype FEMBL150036, 132.5mm SL, Zhanjiang, Guangdong Province, China, collected by Jiaguang Xiao, January 2016.

Paratypes FEMBL150015–150019, FEMBL150031– 150035, FEMBL150037–150050, FEMBL150109–150123, FEMBL150131–150178, 87 individuals, 95.0–166.0mm SL, collection data in consonance with holotype; FEMBL 2705–2736, 32 juvenile individuals, Zhanjiang, Guang- dong Province, collected by Xiongbo He, December 2015; FEMBL150124–150130, 7 individuals, 85.0–138.0mmSL, an aquaculture base in Yinhai District (21˚26´ 27.52´´N, 109˚17´7.72´´E), Beihai, Guangxi Province, collected by Ting Guo, September 2017.

Etymology The name ‘parasihama’ indicate the close re- semblance toin external morphology.

Diagnosis Dorsal fin rays XI–XII (mostly XI), I+(18– 21) (mostly 20); anal fin rays II+(19–21) (mostly 20); la- teral line scales 65–70, scales above lateral line 4–5; gill rakers (2–3)+(5–7); vertebra: abdominal 14, modified 4–7, caudal 13–16, and total 34 (Table 2). Body without dark blotches or mid-lateral stripe; dorsal fins without se- veral rows of dark spots on rays; anal fin without tiny darkspots. The anterolateral extensions of swim bladder are uni- que with some dendritic or sometimes stunted blind tubule, unilateral and outward, anterolateral extensions extending along the abdominal and about one-third to half of the body of swim bladder in length.

Table 2 Morphometric measurements and counts of Sillagoparasihamasp. n.

Description General body features are shown in Fig.1. Counts and measurements are given in Table 2. The des- cription is based on holotype, data of paratypes are shown in parentheses.

Body elongate, abdomen wide, body shape curved, an- terior slightly pyramidal, posterior cylindrical; anterodor- sal profile smooth. Head large, its length 25.9% (18.4%– 29.0%) in SL. Snout long, its length 43.3% (38.9%– 48.0%) of HL. Eye moderate, its margin slightly covered with the adipose eyelid, diameter 20.0% (14.0%–37.1%) of HL. Interorbital region flat, width 6.2% (3.5%–11.1%) of HL. The anterior ocular of eye has two nostrils with a one-way anterior flap on posterior border of anterior nos- tril and a lack of flaps on posterior nostril. Anterior nostril slightly larger than posterior nostril. Mouth small, termi- nal, tips of jaws almost on samevertical. Jaws with 2–3 rows of small canines forming a wide tooth band gradual- ly becoming narrower posteriorly, ending in one row. Pa- latine and tongue toothless. Vomer with 3–4 rows of ca- nine teeth. Posterior margin of preopercle weakly serrated. Gill aperture large, lateral, extending to the pelvic side of the head, stopping at the middle bottom of opercle. Gill rakers on first arch pointed, short, and sparse. Caudal pe- duncle short, its depth 77.2% (52.7%–95.7%) of its length.

Scales deciduous, size moderate, ctenoid but cycloid on prenasal area, cheek and the lower part of pre-opercular- mandibular canal. Pectoral fin base and pelvic fin base lacking scales. The lateral line begins above gill aperture and anterior portion of the pectoral fin, extending along thecurve of dorsal edge to the end of body.

Dorsal-fin rays XI (XI–XII), I+20 (18–21), with blurry black spots on membrane. Anal-fin rays II, 19 (19–21), not extending to caudal fin origin when placed flat. Pec- toral-fin rays 17 (16–17), slender. Pelvic fins broad, I, 5, roughly triangular, and shorter than pectoral fin. Caudal- fin rays 17 (16–18), tail fork shallow. Comparisons of me- ristic characters of twelvespecies with two poste- rior extensions of the swim bladder are shown in Table 3.

Table 3 Comparison of Sillagoparasihamasp. n. and other 11 species of Sillago with two posterior extensions of the swim bladder

Notes: a, this study; b, McKay (1985, 1992); c, Panhwar. (2018); d, Xiao. (2016); e, Kaga. (2010); f, Gao. (2011); g, Golani. (2014); h, Kaga and Ho (2012); i, Xiao (2018).

Color of fresh specimens Head surface bright-brown and trunk light yellowish-brown, grading to silver on ab- domen. Dorsal side of snout brownish-gray. Cheek slight- ly silver, little black dots massed on the anterior inferior part of eyes, the color dark-brown. Body without stripes. The anterior half of ventral side sometimes with some dark pigments. Dorsal fins yellowish-hyaline, with small dark spots on fin membrane. Pectoral, pelvic, and anal fins yel- lowish-hyaline without dark spots. Caudal fin yellowish dusky with grayish-brown margin posteriorly.

Swim bladder Swim bladder large, covered by a thick layer of fat; with two anterior extensions extending to the anterior part connected with the occipital bone, two dum- py posterior extensions penetrating into caudal region witha large gap between them, one usually longer than the other.Two anterolateral extensions originate from anteriorly, each branch into anterior and posterior sub-extensions: the an- terior one comprising a short, simple blind tubule and some-times absent. The posterior sub-extensions are unique with some dendritic or sometimes stunted blind tubule, unila- teral and outward, extending along the abdominal and about one-third to half of the body of swim bladder in length. A single duct-like process originating from the pelvicsur- face of swim bladder extending to the urogenital opening(Figs.2a, 3a).

Fig.2 Swim bladders of S. parasihama sp. n. (a) and S. si- hama (b).

Otolith Otoliths shape fusiform; most of the edge is smooth, with only slight protuberances at the posterior end. There is a main groove throughout the otolith, with an open- ing at both ends. The surface of the no-groove side is bright,with radial stripes and tumor-like rimes in the middle. There are distinct protuberances on the pelvic edge. There is no significant difference between the left and right otoliths (Fig.4).

Distribution and habitatis present- ly known only from the southern coast of China (Beihai and Zhanjiang).It may distributes along the beaches, sand- bars, and the mangrove creeks of sandy substrate. It is ge- nerally mixed cultured with other fish or shrimp in open- air ponds (usually 1.6m in deep).The species prefer to dive into the soft sand bottom of ponds.

Fig.3 Swim bladders of 10 Sillago species with two posterior extensions. (a), S. parasihamasp. n.; (b), S. sinica; (c), S. si- hama; (d), S. shaoi; (e), Sillago sp.; (f), S. indica; (g), S. parvisquamis; (h), S. intermedius (McKay, 1992); (i), S. caudicu- la Kaga etal. (2010); (j), S. suezensissketch based on Golani etal. (2014).

Fig.4 Otoliths of Sillagoparasihamasp. n. (a), left otolith’s concave view; (b), left otolith’s convex view; (c), right otolith’s concave view; (d), right otolith’s convex view. Scale bar, 1000μm.

Comparison According to the subgenera-grading sys- tem in genusproposed by McKay (1985),.belongs to subgenus(). We confirm- ed the validity of.by comparing it with all species in this group. Among the elevenspecies with two posterior extensions,.can be easi- ly distinguished from.and.by the body coloration (dusky black blotches are present on the body of.and.); from.and.by the dusky spots on the second dorsal fin membranes (five or six rows in.and three or four rows in.). We could also distin- guish.from.,.,andsp. by the coloration of anal fin (the anal fin ofis hyaline without black spots, but the anal fins of,andsp. yellowish, interradial mem- branes with black dots). Moreover, we could also distin- guish.from.,.,.andsp. by the faint midlateral stripe on the body of them.

Moreover, we could also distinguish most of these spe- cies based on the meristic and morphometric characters in Table 3..is easily distinguishable from others by the following characteristics:.by hav- ing a smaller head (18.4%–29.0% SL in.. 33.0% in.) and fewer soft rays in anal fin (19–21 in.. 23 in.); from.by having fewer scales above lateral line (4–5 in.. 6–7 in.) and a smaller head (18.4%–29.0% SL in..30.0%–31.0% in); from.,.,.and.by having 34 total ver- tebrae (35 in., 39–40 in., 35–36in.and 37–39 in.); from.and.by having 65–70 scales on lateral line (79–84 in.and 75–79 in.), from.by having fewer scales above/be- low lateral line (4–5/8–10 in.. 7/(11–12) in.); from.by having fewer gill rakers (2–3/5–7 in.. 3/8–9 in.); from.by having fewer soft rays in dorsal fin (18–21 in.. 22–23 in.) and soft rays in anal fin (19–21 in.. 23–24 in.); from.and.by having fewer gill rakers (2–3/5–7 in.. 4/11 in.and 3–4/8–10 in.).

.is most similar with.in the countable characters and color pattern. However, their swim bladders are obviously different (Figs.2, 3). The antero- lateral extensions of swim bladder ofare unique with some dendritic or sometimes stunted blind tu- bule, unilateral and outward, anterolateral extensions ex- tending along the abdominal and about one-third to half of the body of swim bladder in length; but the anterola- teral extensions of swim bladder ofare kinky, long and complicated, and then extending along the abdo- minal wall below the peritoneum to the base of posterior extensions. By contrast,is more similar to.on swim bladder whose anterolateral extensions of swim bladder also with some stunted blind tubule. But this structure is usually more complicated in., and the posterior sub-extensions of swim bladder of.extending along the abdominal and about one- third to half of the body of swim bladder in length.

Genetic analysis of thegene Forty-three sequen- ces of 9species were used in the genetic analysis. The genetic distances amongspecies ranged from 0.084 to 0.231, and the intraspecific distances ranged from 0.000 to 0.004 (Table 4). The NJ tree based ongene sequences (Fig.5) revealed that all the 9 species formed exclusive lineages.

4 Discussion

The new species would be identified as.ac- cording to the countable characters and color pattern (Mc- Kay, 1992). However,.is clearly different from.in the shape of the swim bladder (see Com- parison part). The swim bladder of.is si- milar to.. But.is more complex, and can be distinguished fromby the differences on counts of lateral line scales and vertebrae, as well as the color of the second dorsal fin. In addition,is allopatric with.which occurs from the Bohai Sea to the Taiwan Strait (Gao., 2011)..was described based on holotype only. Unfortunate- ly, the holotype may have been lost (McKay, 1985), and its swimbladder structure and vertebral count are unknown. However,.still can be clearly distinguish- ed from.by obviously larger head (Table 2)..is another species we did not examine any specimens. But this species is different within color pattern and scale counts above/below lateral line (see Comparison part), and in genetics.

Table 4 Net genetic distances (K2P) within (on the diagonal) and between (below the diagonal) the 9 Sillago species

Fig.5 Neighbor-joining (NJ) tree using COI sequences of 9 species of Sillago. The NJ tree was constructed under the K2P model using Sillaginodespunctata as outgroup. Bootstrap support values of >50% from 1000 replicates are shown.

The genetic distances between.and otherspecies ranged from 0.159 to 0.231, much largerthan intraspecific genetic distances, strongly supporting the validity of the new species. The phylogenetic analysis re- covered nine exclusive lineages representing nine validspecies..was grouped with the clade including.,., and..Among genus,exhibits high genetic diversity in Indo-West Pacific. A number of recently describedspecies across Indo-West Pacific–.(from Omanand Madagascar),.(from China),.(from the northern Red Sea and Mediterranean),.(from Taiwan Strait) and.(from the northern ArabianSea)–have been ever misidentified as.(Kaga., 2010; Gao., 2011; Kaga and Heemstra, 2013; Golani., 2014; Xiao., 2016; Panhwar., 2018).is a new one isolated from. Thus, a thorough taxonomic revision ofspecies from Indo-West Pacific is necessary.

A synopsis of Sillaginidsfrom China is presented ba- sed on this study and previous records (McKay, 1985, 1992; Gao., 2011; Xiao., 2016). Presently, a total of 37valid species of Sillaginidae are recognized. Fourteen of them have been recorded from China, including.,.,.,.,.,.,.,.,.,.,.,.,sp. and.(Mc- Kay, 1985, 1992; Gao., 2011; Xiao., 2016; Xiao, 2018). Among them,.,.,., andsp. are identified from the Taiwan Strait to the South China Sea;.is widely distributed from the south of Bohai Sea to the South China Sea;.dis- tributes from the Bohai Sea to the Taiwan Strait;.is currently sampled from the Taiwan Strait;.and.are found from the northern coast of China;.,.,.have only beenrecorded from Taiwan;.and.have been described in Hainan and the Beibu Gulf, re- spectively (Xiao, 2018). We also provided a distribution map (Fig.6) and a key characteristic of the 11spe- cies with two posterior extensions on swim bladder, which should be particularly helpful for species identification and further taxonomic study of Sillaginids.

Fig.6 Distribution map of the 11 Sillago species with two posterior extensions on swim bladder according to McKay (1992), Xiao (2018), and this study.

Key to the 11species with two posterior extensions on swim bladder

1. Body with dark spots .............................................................................................................2

- Body uniform in color............................................................................................................ 3

2. Anal fin rays 23–24; vertebrae 35–36........................................................

- Anal fin rays 21–22; vertebrae 34............................................................

3. HL/SL usually less than 33%................................................................................................. 4

- HL/SL 33%............................................................. .............................

4. Membrane of the second dorsal fin with several rows of dusky spots...................................5

- Membrane of the second dorsal fin without dusky spots.......................................................6

5. The second dorsal fin with 5 or 6 rows of dusky spots along rays........

- The second dorsal fin with 3 or 4 rows of dusky spots along rays....................

6. Membrane of anal fin with spots............................................................................................7

- Membrane of anal fin hyaline................................................................................................8

7. Lower gill rakers 7–8; anterolateral extensions of swim bladder thin................

- Lower gill rakers 5–6; anterolateral extensions of swim bladder strong............

8. Body with a faint mid-lateral dark stripe......................................................

- Body without mid-lateral dark stripe..................................................................................... 9

9. Preopercle and most of the opercle without scales.......................................

- Preopercle and most of the opercle with scales.................................................................... 10

10.The anterolateral extensions of swim bladder with dendritic or sometimes stunted blind tubule, extending to one-third to half of the swim bladder .....................

- The anterolateral extensions of swim bladder kinky and complicated, extending to the base of posterior extensions.....................................................................................

Acknowledgements

We are very grateful to Mr. Jianwei Zou for help in sam- ple collection. This work is supported by the National KeyResearch and Development Program of China (No. 2019 YFD0901301), and the National Natural Science Founda- tion of China (Nos. 41976083, 41776171).

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May 5, 2021

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(Edited by Qiu Yantao)